The integral membrane enzyme PagP alternates between two dynamically distinct states
The integral membrane enzyme PagP alternates between two dynamically distinct states
复制标题
DOI：
10.1073/pnas.0402324101
复制
发表时间：
2004-06-29
影响因子：
11.1
通讯作者：
Kay, LE
中科院分区：
文献类型：
--
作者：
Hwang, PM;Bishop, RE;Kay, LE
PhoPQ激活基因P（PagP）是革兰氏阴性菌中将sn-1棕榈酸链从磷脂转移到脂多糖的完整膜酶。最近的X射线晶体学研究确定，sn-1棕榈酸酯结合在PagP 13桶中心的长腔中。允许底物进入桶的中心核所需的高流动性与在外围环中组装明确限定的结构的需要形成对比，其中许多关键催化残基位于外围环中。为了深入了解动力学如何与PagP的功能相关，将该酶重组为CYFOS-7，一种支持酶活性的洗涤剂。在这些条件下，PagP存在于两种状态（放松（R）和紧张（T））之间的平衡。用H-1-N-15 NMR谱研究了交换的动力学和热力学，R->T跃迁的Δ H = -10.7 kcal/mol，Δ S = -37.5 cal/mol-K。两种状态之间的化学位移的比较表明，主要的结构变化发生在大的细胞外L1环和相邻区域的β桶。除了R，T相互转换，其他构象交换过程中观察到的R状态，显示它是相当灵活的。因此，出现了这样一幅图景：R态的迁移性促进了底物的进入，而相对刚性的T态在蛋白质的一个已知对催化至关重要的区域采用了完全不同的构象。在动态不同状态之间切换的能力可能是PagP的催化循环的关键特征。
PhoPQ-activated gene P (PagP) is an integral membrane enzyme that transfers the sn-1 palmitate chain from phospholipid to lipopolysaccharide in Gram-negative bacteria. A recent x-ray crystallographic study established that the sn-1 palmitate binds within a long cavity at the center of the PagP 13 barrel. The high mobility required to permit substrate entry into the central core of the barrel contrasts with the need to assemble a well defined structure in the peripheral loops, where many key catalytic residues are located. To gain insight into how dynamics relate to the function of PagP, the enzyme was reconstituted into CYFOS-7, a detergent that supports enzymatic activity. Under these conditions, PagP exists in equilibrium between two states, relaxed (R) and tense (T). The kinetics and thermodynamics of the interchange have been investigated by H-1-N-15 NMR spectroscopy, with DeltaH = -10.7 kcal/mol and DeltaS = -37.5 cal/mol-K for the R-->T transition. A comparison of chemical shifts between the two states indicates that major structural changes occur in the large extracellular L1 loop and adjacent regions of the beta barrel. In addition to the R,T interconversion, other conformational exchange processes are observed in the R state, showing it to be quite flexible. Thus a picture emerges in which substrate entry is facilitated by the mobility of the R state, whereas the relatively rigid T state adopts a radically different conformation in a region of the protein known to be essential for catalysis. The ability to switch between dynamically distinct states may be a key feature of the catalytic cycle of PagP.